B lymphocytes play a critical role in host defenses; however critical mechanisms of their activation and differentiation are poorly understood. For example, intracellular Ca2+ is recognized as being central, yet relatively little is known about how it is regulated. Much of what we know about Ca2+ in lymphocytes has been learned from studies of T cells. For example, Ca2+-Release Activated Calcium (CRAC) channels, which are believed to be their major, and possibly only, antigen receptor-operated Ca2+ channel in T cells, has been studied in great detail, but it has not yet been described in primary B cells. In fact, limited previous studies and our preliminary data demonstrate that the mechanisms of Ca2+ signaling in B cells are more complex than described for T cells, and whether CRAC channels play a similar indispensable role in B cell immunological functions now seems unlikely. We show that B cells utilize CRAC channels, but also additional Ca2+-permeant Non-Selective Cation Channels (NSCC). We further demonstrate that these NSCCs are activated by stimuli associated with innate immune responses including viral and bacterial agonists of Toll like receptors (TLR) called Pathogen-Associated Molecular Patterns (PAMPs), and by mechanical forces. These responses are not observed in T cells. We speculate that these dissimilarities between B and T cells reflect the fundamental difference in the way that these two lymphocyte populations recognize antigens. B cells recognize antigens directly through contact with immunoglobulin molecules at their surface. In contrast, antigen recognition by T cells is indirect and depends upon permissive interactions with antigen presenting cells (APC's). As a safeguard against autoimmunity, recognition of antigen alone is not sufficient to trigger activation; a second signal indicating the presence or absence of danger - TLR ligands such as double-stranded RNA, LPS, LTA, unmethylated CpG DNA that are unique to pathogens - is required. Much of the decision making for a T cell is made by the APC, while the B cell is on its own in terms of integrating costimulatory information and this will require the more complex signaling that we have uncovered. One of the central underlying ideas of this application, therefore, is that appropriate responses of B cells will reflect the coordinated activation of distinct Ca2+ permeant (NSC and CRAC) channels by the overlapping pathways activated by the BCR and innate stimuli. We will use single cell patch clamp recording and calcium imaging methods to biophysically define these channels activated by BCR and innate stimuli, but also a novel modification of this approach to unravel the complex intracellular signaling pathways that coordinate their activation and functions. Our results will have fundamental importance for understanding the basis of immune competence, immune deficiency, and autoimmunity.